Background

As a tool for genetic research and breeding, genetic linkage maps have been widely used to discover the position and to clone genes controlling biotic and abiotic stress resistance, agronomic and seed quality traits and to facilitate marker-assisted selection of the traits with low heritability and/or high phenotyping cost. In soybean, the first molecular genetic linkage map was reported in 1990 [1]. The map contained 150 restriction fragment length polymorphism (RFLP) markers that were mapped using an F2 population with 60 progeny derived from a cross of A81-356022 (G. max) × PI468916 (G. soja). Subsequently, a map with 130 RFLPs was constructed based on an F2 population with 69 progeny from a cross of Minsoy × Noir 1 [2], and a map with 165 RFLPs, 25 random amplified polymorphic DNA (RAPD) markers and 650 amplified fragment length polymorphisms (AFLPs) based on 300 recombinant inbred lines (RILs) from PI437654 × BSR101 [3] were reported. The early genetic linkage maps were primarily based on RFLP or AFLP markers and due to the lack of polymorphism or the complexity of the multiple banding patterns with these markers, simple sequence repeat (SSR) or microsatellite markers were proposed and then evaluated for the construction of genetic linkage maps [4, 5]. Cregan et al. (1999) [6] developed three separate linkage maps containing a total of 1421 markers including 606 SSRs, 689 RFLPs, 79 RAPDs and 47 other markers. These markers were mapped using three RIL populations: the Minsoy × Noir 1 population with 240 RILs, the A81-356022 × PI468916 population with 57 F2 plants, and the Clark × Harosoy population with 59 F2 plants and resulted in 20 linkage groups which were assumed to correspond to the 20 pairs of soybean chromosomes. Song et al. (2004) [7] constructed an integrated soybean linkage map using the three mapping populations used by Cregan et al. (1999) [6] as well as two additional mapping populations from Minsoy × Archer with 233 RILs, and Archer × Noir 1 with 240 RILs. The consensus map contained 1849 markers including 1015 SSRs, 709 RFLPs, 73 RAPDs and 52 other markers [7]. As large numbers of expressed sequence tags (ESTs) and genomic sequence became available in later years, Choi et al. (2007) [8] discovered >5500 single nucleotide polymorphism (SNP) markers by comparing DNA sequences acquired from a set of diverse genotypes after PCR amplification and sequence analysis of the EST or genomic sequences. A total of 1141 of the 5500 SNPs were mapped using three mapping populations including the Minsoy × Noir 1 with 164 RILs, Minsoy × Archer with 89 RILs as well as the Evans × PI 209332 with75 RILs [8]. Hyten et al. (2010) [9] added 2651 additional SNPs to the linkage maps created by Choi et al. (2007) [8] using the same Minsoy × Noir 1, Minsoy × Archer and Evans × Peking populations [9]. All of the molecular markers on these linkage maps were developed before the soybean whole genome sequence was available, thus, the markers were not evenly distributed and did not sufficiently cover all of the genomic regions of the soybean genome with a total sequence length >1100 Mb [10].

The Williams 82 Glyma1.01 whole genome sequence was completed and published in 2010 [11]. The genome sequence is widely used for the study of gene structure [12–14], syntenic relationships among legume species [15–18], identification of genes [19–22], the development of additional molecular markers and for other uses. Song et al. (2013) [23] identified 209,903 SNPs by mapping short reads from each of eight soybean accessions which included six cultivated (Glycine max (L.) Merr.) and two wild soybean (G. soja Sieb. & Zucc.) genotypes and selected 60,800 SNPs for the design of the SoySNP50K Illumina Infinium BeadChip [23]. The BeadChip has been successfully used to genotype the entire USDA Soybean Germplasm Collection containing 19,652 accessions including 1168 wild and 18,484 cultivated soybean accessions [24], the dataset is available at Soybase, the USDA, ARS Soybean Genetics and Genomics Database, http://www.soybase.org/snps/download.php) and is being used for genome-wide association analysis [25–29], quantitative trait loci (QTL) analysis [26], genetic diversity analysis and the identification of regions associated with domestication and selection imposed by modern breeding. In addition, Song et al. (2010) [30] identified a total of 210,990 SSRs with di-, tri-, and tetranucleotide repeats of five or more in the soybean whole genome sequence which included 61,458 SSRs consisting of repeat units of di- (≥10), tri- (≥8), and tetranucleotide (≥7), and developed a database (BARCSOYSSR_1.0) of locus-specific SSR markers with a high likelihood of polymorphism. A database with the primer sequences and their genome positions for 33,065 SSRs in the Glyma1.01 assembly was created [30]. The database also included the physical positions of 3322 SNPs in the Glyma1.01 build, which were mapped by Hyten et al. (2010) [9]. These molecular markers plus the markers developed previously [6–9, 31–35] are being used by researchers in the soybean community. However, with the updating of the Glyma1.01 build based on the high-resolution linkage maps resulting from this research, the genome positions of these markers need to be redefined.

The Glyma1.01 build captured approximately 975 Mb of sequence across the 20 chromosomes. The Glyma1.01 whole genome sequence contained 236 unanchored scaffolds with lengths ranging from 10 to 100 kb and 51 unanchored scaffolds with lengths greater than 100 kb. The assembly was basically built based on the integrated linkage maps [7, 9] and a genetic map with additional markers specifically selected to aid in the pseudomolecule assembly [36]. However, the marker density on any one of these linkage maps was still insufficient to fully cover all regions of the soybean genome. In addition, the number of RILs genotyped for the construction of the previous linkage maps was relatively small (60–240 RILs) [7, 9]. These deficiencies may result in low resolution, large gaps, and incorrect marker order in the linkage maps, and in turn, may cause incorrect orientation or misplacement of scaffolds in the Glyma1.0 whole genome sequence assembly of soybean.

The objectives of this research were to construct high resolution linkage maps using large mapping populations, to identify misplaced or incorrectly orientated genomic regions, to anchor additional scaffolds in the Glyma1.01 assembly, and to position SSR and SNP markers in the Wm82.a2.v1assembly.

Results

Construction of high resolution linkage maps

A total of 23,814 SNPs were polymorphic among 1083 RILs in the Williams 82 x PI479752 (WP) and 17,150 SNPs among the 922 RILs in the Essex x Williams 82 (EW) population. After elimination of SNPs with missing >10 % or segregation distortion significant at the 1 % level based on χ 2 tests, 21,478, 11,922 and 27,431 SNPs were mapped in the WP, EW and WP + EW populations, respectively. The number of mapped SNPs in each linkage group ranged from 825 to 1910 in the WP, 132–1313 in the EW and 938–2481 in the WP + EW populations. The total genetic linkage map distance was 2445.8 cM in the WP and 2647.6 cM in the EW population (Table 1 and Additional file 1: Table S1).

Table 1 Number of SNPs mapped to each linkage group, linkage group length based on the Williams 82 × PI479752 (WP) and the Essex × Williams 82 (EW) populations and the number of SNPs common to the two populations and SNPs unique to one population
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Identification of misassembled genomic regions or anchorable scaffolds in Glyma1.01

Of the 21,478 SNPs mapped in WP and 11,922 in EW, 5969 SNPs were present in both populations and the number of common SNPs per chromosome ranged from 67 on chromosome Gm14 to 742 on chromosome Gm18. Marker order on the genetic linkage maps was used to identify major genomic regions of the Glyma1.01 that required reorientation and/or re-positioning. Analysis showed that the order of the common markers on the two linkage maps was highly consistent and the order of the SNPs was generally consistent with their physical positions along the corresponding chromosomes of Glyma1.01 (Additional file 2: Figure S1). However, there were 22 regions or single markers that required re-positioning or reorientation based upon marker orders supported by both the WP and EW mapping data (Table 2). In these regions, the SNP markers had consistent order along the linkage maps in both mapping populations but their order was not consistent with physical position in Glyma1.01. For example, there were regions on Gm04, Gm05, Gm10 and Gm13 where the order of SNPs on both linkage maps was identical, but the order of those SNPs in the Glyma1.01 assembly was reversed (Additional file 2: Figure S1). In addition, a number of individual markers or sets of markers identified sequence that was placed on the wrong chromosome (Table 2). There were a total of 15 regions that required re-positioning or reorientation based upon marker orders available from either the WP or EW mapping data (Table 2). In addition, 28 unanchored scaffolds with a total length of 3.6 Mb in Glyma1.01 were anchored as a result of markers in either the WP or EW map, or both, that defined the scaffold genome position (Table 3).

Table 2 Regions or single markers in Glyma1.01 that required re-positioning or reorientation based upon marker orders supported by both or either the W82 x PI79752 and/or the Essex x W82 mapping data
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Table 3 Twenty-eight previously unanchored scaffolds for which there were markers in either the Williams 82 × PI479752 (WP) or the Essex × Williams 82 (EW) map, or both, to define their genome position
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The Wm82.a2.v1 assembly

Based on the two dense linkage maps and additional analyses, sequence breaks from Glyma1.01 were identified and reassembled. The new build of the 20 chromosomes captured 949.2 Mb. The total sequence including the 1170 unmapped scaffolds was 978.5 Mb. The plots of the genetic on physical distance of the SNPs in Glyma1.01 and Wm82.a2.v1 showed that major regions, such as on Gm05 and Gm13 with an inconsistent order of SNPs on linkage maps vs. physical position in the Glyma1.01 build were corrected in the Wm82.a2.v1 assembly (Additional file 3: Figure S2). Further comparison of the physical positions of the SNPs in Glyma1.01 vs. Wm82.a2.v1 showed that sequence assembly errors in the regions indicated in Table 2 and Table 3 were all corrected in Wm82.a2.v1 (Additional file 1: Table S1). In addition, a total of 28 scaffolds with mapped SNP markers were anchored to the new build. The new assembly which is completed at the Department of Energy, Joint Genome Institute is available at http://www.phytozome.jgi.doe.gov/pz/portal.html.

Based upon the plots of genetic distance on physical distance of the SNPs in Wm82.a2.v1 and mapped in either the WP or EW population, the regions with high and low recombination rate were defined. These plots allowed the delimitation of the approximate positions of euchromatic and heterochromatic regions along each chromosome (Table 4). The two regions covered approximately 43 % and 47 % of the total estimated genome sequence, respectively.

Table 4 Approximate positions of heterochromatic and euchromatic regions in the Wm82.a2.v1 whole genome sequence
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Positions of commonly used markers in the Wm82.a2.v1 assembly

Of the 33,065 SSRs and 3322 SNPs in the BARCSOYSSR_1.0 database, 32,602 SSRs and 3314 SNPs were unambiguously positioned in the Wm82.a2.v1 assembly. A total of 2122 SNPs and 7092 SSRs were in the genes defined in Wm82.a2.v1 and the total number of unique genes in which these SSR and SNP markers resided was 7686 (Additional file 4: Table S2).

Among the 60,800 SNPs originally selected for inclusion in the SoySNP50K BeadChip [23], 60,556 SNPs were positioned in the new assembly and a total of 20,271 SNPs were in 14,880 different genes. The positions of 244 SNPs in the Wm82.a2.v1 assembly could not be determined (Additional file 1: Table S1).

Discussion

The two linkage maps created in this study have the highest density of markers and are based on the largest number of recombinant inbred lines that have been reported in soybean to date. Simulation studies indicated that a low number of RILs in a population frequently caused inversions of marker order and breakage in linkage groups and that the precision of the maps is highly dependent on the number of RILs [37]. For the purpose of integrating large numbers of markers into a linkage map, the WP population which was derived from the cross of cultivated by wild soybean accessions was developed. The large genetic divergence between the two subspecies allowed us to identify and map large numbers of SNPs in a single population. One concern with the linkage maps from G. max x G. soja was the possibility of paracentric inversions and reciprocal translocations between the cultivated soybean and certain wild soybean accessions [38, 39]. However, we did not observe such regions in the linkage maps of WP based on the order of approximately 6000 common SNPs mapped in both the WP and EW populations.

Besides the number of markers and size of the RIL populations, utilization of evenly distributed markers across the whole soybean genome was also essential to ensure maps with high resolution. The SNPs in the SoySNP50K BeadChip were carefully selected in order to equalize the distance between selected SNPs in the euchromatic and heterochromatic regions along each chromosome and the BeadChip was able to generate high quality genotyping data [23]. The resulting two linkage maps had better coverage and higher resolution than any other soybean linkage maps reported previously. The high quality of the two linkage maps is supported by the very consistent order of the common markers in the two maps.

Even though the Glyma1.01 build was well constructed, we identified regions where the marker physical order was inconsistent with the WP and EW linkage maps. Most of these regions either had insufficient marker numbers or lacked markers with recombination in the previous linkage maps [7, 9, 36] on which the Glyma1.01 assembly was based. The misassembled or improperly oriented regions identified by our linkage maps covered all of the major regions reported by Lee et al. (2013) [40] and the regions were moved or re-assembled in the Wm82.a2.v1 assembly. Of course, refinement of some regions may still be required especially in the heterochromatic regions where limited recombination was observed.

In order to determine the approximate positions of the euchromatic and heterochromatic regions of the genome, the cumulative genetic distances (cM) were plotted against their corresponding cumulative physical distance (Mbp) via the mapped SNP loci positions on the genetic linkage map and their genome sequence position along each chromosome. The region between the two inflection points of the cumulative genetic distance against cumulative physical distance on the plot was defined as the heterochromatic region [23]. The reliability of defining heterochromatic regions using this method was validated by the conventional 4,6-diamidino-2-phenylindole dihydrochloride staining method in rice [41].

Because of the many reports of genes/QTL in the soybean genome positioned using SSR or SNP markers, the corresponding physical position of the molecular markers in the new assembly vs. the older assembly is frequently requested by users. We identified physical positions for almost all of the markers in the BARCSOYSSR_1.01 database and the SoySNP50K BeadChip in the Wm82.a2.v1 vs. the Glyma1.01 assemblies. The updated information is anticipated to facilitate the identification of molecular markers in desired positions of the genome and make the SSR and SNP databases more user-friendly.

Conclusions

Two high density genetic linkage maps of soybean based on 21,478 SNP loci mapped in the G. max x G. soja population with 1083 recombinant inbred lines and 11,922 SNP loci mapped in the G. max x G. max population with 922 RILs were constructed. The maps contained the highest number of markers and were constructed based on the largest mapping populations in soybean to date. With the high density genetic linkage maps, false joins or mis-placed scaffolds and unanchored scaffolds in the first version of the soybean whole-genome sequence assembly (Glyma1.01) were identified and the corresponding scaffolds were broken or reassembled to a new Wm82.a2.v1 assembly which is available at the site http://www.phytozome.jgi.doe.gov/pz/portal.html/ of the Department of Energy, Joint Genome Institute. In addition, the euchromatic and heterochromatic regions along each chromosome of the soybean were delimited and the positions of commonly used soybean SSR and SNP markers were determined based on the Wm82.a2.v1 assembly. The information will facilitate the genetic and genomics research in soybean.

Methods

Mapping populations

A cross between cultivated soybean (Glycine max L. Merr.) Williams 82 and wild soybean (G. soja Sieb. et Zucc.) PI479752 (WP) was made at Beltsville, MD. The WP population consists of 1083 F5-derived RILs. The Essex × Williams 82 population with 922 F5-derived RILs was developed at the University of Tennessee, Knoxville, TN. One of the parents in both mapping populations was Williams 82, which is the cultivar that was used in the synthesis of the first whole-genome sequence of the soybean provided in the Glyma1.01assembly [11].

Genotyping RILs of the mapping populations with the SoySNP50K BeadChip

Song et al. (2013) [23] identified 209,903 SNPs by mapping short reads from each of eight soybean accessions which included six cultivated and two wild soybean genotypes and selected 60,800 SNPs for inclusion in an Illumina Infinium BeadChip that ultimately contained more than 52,000 SNPs. The SNPs for the SoySNP50K BeadChip were selected so as to equalize the distance between selected SNPs in the euchromatic and heterochromatic regions, increase assay success rate, and minimize the number of SNPs with low minor allele frequency. Of the 60,800 SNPs selected for the BeadChip, 50,701 were targeted to euchromatic regions and 10,000 to heterochromatic regions of the 20 soybean chromosomes. In addition, 99 SNPs were targeted to unanchored sequence scaffolds. The BeadChip was used to genotype the RILs in the WP and the EW populations using the Illumina platform following the Infinium® HD Assay Ultra Protocol (Illumina, Inc. San Diego, CA) and the SNP alleles were called using the GenomeStudio Genotyping Module v1.8.4 (Illumina, Inc. San Diego, CA) as described previously [23].

Construction of the high-density linkage maps

Linkage maps for the WP and EW populations were created using the MSTMap software [42] and the genetic distance between SNPs was calculated using JoinMap 4.0 [43]. Before linkage map analysis, loci with segregation distortion in the population (p < 0.01) or with missing data >10 % were eliminated. RILs with missing data >10 % were also removed. In order to reduce the time required to determine the order and genetic distance of the SNPs in each linkage group, SNPs with identical allele segregation patterns among RILs of WP or EW populations were clustered into groups, and only one SNP from each group was included in linkage analysis. The remaining SNPs were assigned to the same linkage map position as the representative SNP after completion of the linkage analysis. A LOD of 11 was used to cluster the markers into linkage groups. Recombination values were converted to genetic distances using the Kosambi mapping function [43].

Identification of genomic regions in the Glyma1.01 assembly that required re-positioning or reorientation

Genetic linkage map positions of SNPs on the linkage maps of WP and EW were compared with their physical positions in the Glyma1.01assembly and regions that required re-positioning or reorientation were identified based upon marker orders supported by the WP and/or the EW mapping data and scaffolds with false joins were broken and re-assembled. The physical positions of these SNPs in the Glyma1.01 were previously reported by Song et al. (2013) [23].

Physical positions of commonly used SSR and SNP markers in the new assembly- Wm82.a2.v1

The BARCSOYSSR_1.0 database consists of 3322 SNPs and 33,065 SSRs [30], and the SoySNP50K BeadChip contained 52,041 SNPs selected from the soybean genome. In order to position these loci in the Wm82.a2.v1 soybean genome sequence, source sequences of the SSR and the sequences flanking the SNP loci were aligned to the Wm82.a2.v1 soybean sequences using standalone Megablast software (http://www.ncbi.nlm.nih.gov/blast/megablast.shtml) with W = 50, cutoff percentage of alignment = 99 and low complexity filtered. The primer sequences of the SSR loci were mapped to the genome sequence using the standalone software e-PCR (ftp://ftp.ncbi.nih.gov/pub/schuler/e-PCR/). SSR positions were definitively determined if both the source sequences and primer sequences of the SSRs aligned to the same region of the genome sequence with expected e-PCR amplicon length and with the SSR motif between the two primer sequences. High stringency alignment (gap = 0, number of mismatch = 0) with e-PCR of primer sequences to the genome sequence was used to map the primer sequences.

Availability of supporting data

The SNP information is deposited in the dbSNP database of NCBI (ss715578401-ss715639200). The new soybean whole genome sequence assembly (Wm82.a2.v1) which is completed at the Department of Energy, Joint Genome Institute is available at http://www.phytozome.jgi.doe.gov/pz/portal.html/. The remaining data sets supporting the results of this article are included within the article and its four additional files.